Insights into the intracellular localization, protein associations and artemisinin resistance properties of Plasmodium falciparum K13

The emergence of artemisinin (ART) resistance in Plasmodium falciparum intra-erythrocytic parasites has led to increasing treatment failure rates with first-line ART-based combination therapies in Southeast Asia. Decreased parasite susceptibility is caused by K13 mutations, which are associated clinically with delayed parasite clearance in patients and in vitro with an enhanced ability of ring-stage parasites to survive brief exposure to the active ART metabolite dihydroartemisinin. Herein, we describe a panel of K13-specific monoclonal antibodies and gene-edited parasite lines co-expressing epitope-tagged versions of K13 in trans. By applying an analytical quantitative imaging pipeline, we localize K13 to the parasite endoplasmic reticulum, Rab-positive vesicles, and sites adjacent to cytostomes. These latter structures form at the parasite plasma membrane and traffic hemoglobin to the digestive vacuole wherein artemisinin-activating heme moieties are released. We also provide evidence of K13 partially localizing near the parasite mitochondria upon treatment with dihydroartemisinin. Immunoprecipitation data generated with K13-specific monoclonal antibodies identify multiple putative K13-associated proteins, including endoplasmic reticulum-resident molecules, mitochondrial proteins, and Rab GTPases, in both K13 mutant and wild-type isogenic lines. We also find that mutant K13-mediated resistance is reversed upon co-expression of wild-type or mutant K13. These data help define the biological properties of K13 and its role in mediating P. falciparum resistance to ART treatment

Tissue ACE phenotyping in lung cancer.

BACKGROUND:Pulmonary vascular endothelium is the main metabolic site for Angiotensin I-Converting Enzyme (ACE)-mediated degradation of several biologically-active peptides (angiotensin I, bradykinin, hemo-regulatory peptide Ac-SDKP). Primary lung cancer growth and lung cancer metastases decrease lung vascularity reflected by dramatic decreases in both lung and serum ACE activity. We performed precise ACE phenotyping in tissues from subjects with lung cancer. METHODOLOGY:ACE phenotyping included: 1) ACE immunohistochemistry with specific and well-characterized monoclonal antibodies (mAbs) to ACE; 2) ACE activity measurement with two ACE substrates (HHL, ZPHL); 3) calculation of ACE substrates hydrolysis ratio (ZPHL/HHL ratio); 4) the pattern of mAbs binding to 17 different ACE epitopes to detect changes in ACE conformation induced by tumor growth (conformational ACE fingerprint). RESULTS:ACE immunostaining was dramatically decreased in lung cancer tissues confirmed by a 3-fold decrease in ACE activity. The conformational fingerprint of ACE from tumor lung tissues differed from normal lung (6/17 mAbs) and reflected primarily higher ACE sialylation. The increase in ZPHL/HHL ratio in lung cancer tissues was consistent with greater conformational changes of ACE. Limited analysis of the conformational ACE fingerprint in normal lung tissue and lung cancer tissue form the same patient suggested a remote effect of tumor tissue on ACE conformation and/or on "field cancerization" in a morphologically-normal lung tissues. CONCLUSIONS/SIGNIFICANCE:Local conformation of ACE is significantly altered in tumor lung tissues and may be detected by conformational fingerprinting of human ACE

Tissue ACE phenotyping in lung cancer

Background Pulmonary vascular endothelium is the main metabolic site for Angiotensin I-Converting Enzyme (ACE)-mediated degradation of several biologically-active peptides (angiotensin I, bradykinin, hemo-regulatory peptide Ac-SDKP). Primary lung cancer growth and lung cancer metastases decrease lung vascularity reflected by dramatic decreases in both lung and serum ACE activity. We performed precise ACE phenotyping in tissues from subjects with lung cancer. Methodology ACE phenotyping included: 1) ACE immunohistochemistry with specific and well-characterized monoclonal antibodies (mAbs) to ACE; 2) ACE activity measurement with two ACE substrates (HHL, ZPHL); 3) calculation of ACE substrates hydrolysis ratio (ZPHL/HHL ratio); 4) the pattern of mAbs binding to 17 different ACE epitopes to detect changes in ACE conformation induced by tumor growth (conformational ACE fingerprint). Results ACE immunostaining was dramatically decreased in lung cancer tissues confirmed by a 3-fold decrease in ACE activity. The conformational fingerprint of ACE from tumor lung tissues differed from normal lung (6/17 mAbs) and reflected primarily higher ACE sialylation. The increase in ZPHL/HHL ratio in lung cancer tissues was consistent with greater conformational changes of ACE. Limited analysis of the conformational ACE fingerprint in normal lung tissue and lung cancer tissue form the same patient suggested a remote effect of tumor tissue on ACE conformation and/or on "field cancerization" in a morphologically-normal lung tissues. Conclusions/Significance Local conformation of ACE is significantly altered in tumor lung tissues and may be detected by conformational fingerprinting of human ACE.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Tissue Specificity of Human Angiotensin I-Converting Enzyme.

Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood.We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests.Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distingushing tissue-specific ACEs

Observed [M+H]⁺ ions of unglycosylated peptides in the mass spectra of human ACE tryptic digests.

<p>^a Acrylamide adduct on cysteine.</p><p>^b Oxidized methionine.</p><p>^c Contains one or two missed cleavage(s) by trypsin.</p><p>Peptides that contain potential N-glycosylation sites are shown in bold.</p><p>Observed [M+H]⁺ ions of unglycosylated peptides in the mass spectra of human ACE tryptic digests.</p

The structures of N and C domains of ACE with potential glycosylation sites and epitopes for mAbs.

<p>Human N domain structure was based on PDB P2C6N and C domain structure—based on PDB 1O86. The epitopes were marked on the N and C domains according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref027" target="_blank">27</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref031" target="_blank">31</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref041" target="_blank">41</a>]. The positions of the epitopes for some mAbs (12 out of 17) are shown by circles on both sides of domain globule. The potential sites of N-glycosylation, 9 on the N domain and 6 on the C domain, are marked by green; Asn494 on the N domain is not seen while Asn1196 is not present in structure of the C domain. The glycosylation sites which might be differently glycosylated in seminal fluid ACE and lung ACE are shown by arrows. Some amino acid residues are shown by numbers according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref038" target="_blank">38</a>] for orientation.</p

Effect of different additives on mAbs binding to seminal fluid and lung ACEs.

<p>ACE activity immunoprecipitated by 17 mAbs to ACE (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.g001" target="_blank">Fig 1</a>) was presented as a normalized value (“binding ratio”) to highlight differences in immunoprecipitation pattern (“conformational fingerprint”) after adding of tested compounds to purified seminal fluid and lung ACEs with that without additives. <b>(A)</b> Effect of 20% of human heat-inactivated plasma. <b>(B</b>) Effect of 80% 3 kDa filtrate of human citrated plasma. <b>C</b>-<b>D</b>. Effect of bilirubin (150 ug/ml) in the absence (<b>C</b>) or presence (<b>D</b>) of human albumin at 8 mg/ml concentration (which correspond to its concentration in 20% serum). Data are presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.g001" target="_blank">Fig 1</a>.</p

Effect of human plasma, seminal fluid and albumins on mAbs binding to ACEs.

<p>ACE activity immunoprecipitated by 17 mAbs to ACE (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.g001" target="_blank">Fig 1</a>) was presented as a normalized value (“binding ratio”) to highlight differences in immunoprecipitation pattern (“conformational fingerprint”) after adding heat-inactivated human citrated plasma, heat-inactivated seminal fluid, as well as human and bovine albumins to purified seminal fluid and lung ACEs with that without additives. <b>A-B</b>. Effect of 20% of heat-inactivated human plasma (<b>A</b>) and heat-inactivated seminal fluid (<b>B</b>); <b>C-D</b>. Effect of human (<b>C</b>) and bovine (<b>D</b>) albumins at concentrations of 8 mg/ml (similar to albumin concentration in 20% serum). Data are presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.g001" target="_blank">Fig 1</a>.</p

Primary immune response in mice to pure somatic ACE from seminal fluid.

<p>Culture fluids from 670 post-fusion cell populations grown in 96-well plates (primary screening) were anylyzed for the presence of antibodies to seminal fluid ACE and lung ACE in parallel in plate precipitation assay (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.g001" target="_blank">Fig 1</a>). Presence of antibodies to seminal fluid and/or lung ACE was detected in 91 wells by precipitated ACE activity and the data are presented as the ratio of ACE activity precipitation from seminal fluid ACE to that from lung ACE (SF/Lung ratio). Discrimination of these two ACEs by antibodies from these positive wells was observed in a wide range (A). Besides expected antibodies, recognizing both ACEs (C) with SF/Lung ratio in the region from 0.5 to 1.5, we identified significant proportions of antibodies which preferentially recognized seminal fluid ACE (B) with SF/Lung ratio more than 1.5, and antibodies which preferentially recognized lung ACE (D) with SF/Lung ratio less than 0.5, correspondingly.</p

Effect of anti-catalytic mAbs on the activity of pure seminal fluid and lung ACEs.

<p>Pure seminal fluid and lung ACEs (5 mU/ml with ZPHL as a substrate) were incubated with mAbs (10 ug/ml), which are anti-catalytic for the N-domain active center, i2H5 and 3A5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref027" target="_blank">27</a>], and for the C domain active center, 1E10 and 4E3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143455#pone.0143455.ref030" target="_blank">30</a>], of ACE. Residual ACE activity was determined with substrates HHL (<b>A</b>) and ZPHL (<b>B</b>) and is presented as the ratio of ACE activity in the presence of mAbs to that without mAbs. Data are also presented as ZPHL/HHL ratio of ACE activity in the presence of mAbs to that without mAbs (<b>C</b>). Results are the mean ± SD of 2–4 experiments, made in duplicates.</p